title: Estrogen receptor-beta sensitizes breast cancer cells to the anti-estrogenic actions of endoxifen.
abstract: BACKGROUND
  We have previously demonstrated that endoxifen is the most important tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing estrogen receptor-alpha (ERα). However, the relevance of ERβ in mediating endoxifen action has yet to be explored. Here, we characterize the molecular actions of endoxifen in breast cancer cells expressing ERβ and examine its effectiveness as an anti-estrogenic agent in these cell lines.
  
  
  METHODS
  MCF7, Hs578T and U2OS cells were stably transfected with full-length ERβ. ERβ protein stability, dimer formation with ERα and expression of known ER target genes were characterized following endoxifen exposure. The ability of various endoxifen concentrations to block estrogen-induced proliferation of MCF7 parental and ERβ-expressing cells was determined. The global gene expression profiles of these two cell lines was monitored following estrogen and endoxifen exposure and biological pathway analysis of these data sets was conducted to identify altered cellular processes.
  
  
  RESULTS
  Our data demonstrate that endoxifen stabilizes ERβ protein, unlike its targeted degradation of ERα, and induces ERα/ERβ heterodimerization in a concentration dependent manner. Endoxifen is also shown to be a more potent inhibitor of estrogen target genes when ERβ is expressed. Additionally, low concentrations of endoxifen observed in tamoxifen treated patients with deficient CYP2D6 activity (20 to 40 nM) markedly inhibit estrogen-induced cell proliferation rates in the presence of ERβ, whereas much higher endoxifen concentrations are needed when ERβ is absent. Microarray analyses reveal substantial differences in the global gene expression profiles induced by endoxifen at low concentrations (40 nM) when comparing MCF7 cells which express ERβ to those that do not. These profiles implicate pathways related to cell proliferation and apoptosis in mediating endoxifen effectiveness at these lower concentrations.
  
  
  CONCLUSIONS
  Taken together, these data demonstrate that the presence of ERβ enhances the sensitivity of breast cancer cells to the anti-estrogenic effects of endoxifen likely through the molecular actions of ERα/β heterodimers. These findings underscore the need to further elucidate the role of ERβ in the biology and treatment of breast cancer and suggest that the importance of pharmacologic variation in endoxifen concentrations may differ according to ERβ expression.
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1stStreet SW, Rochester, MN 55905, USA.
authors.forename: Xianglin
authors.initials: X
authors.lastname: Wu
authors.forename: Malayannan
authors.initials: M
authors.lastname: Subramaniam
authors.forename: Sarah B
authors.initials: SB
authors.lastname: Grygo
authors.forename: Zhifu
authors.initials: Z
authors.lastname: Sun
authors.forename: Vivian
authors.initials: V
authors.lastname: Negron
authors.forename: Wilma L
authors.initials: WL
authors.lastname: Lingle
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Goetz
authors.forename: James N
authors.initials: JN
authors.lastname: Ingle
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Spelsberg
authors.forename: John R
authors.initials: JR
authors.lastname: Hawse
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology; D020847:Estrogen Receptor Modulators / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000235:genetics / Q000378:metabolism*; D004967:Estrogens / Q000494:pharmacology; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D055503:Protein Multimerization; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: C506487:ESR1 protein, human; D020847:Estrogen Receptor Modulators; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D004967:Estrogens; D013629:Tamoxifen; C055492:4-hydroxy-N-desmethyltamoxifen; D004958:Estradiol; D019389:Cytochrome P-450 CYP2D6